Trans-membrane adenylyl cyclase (tmAC) isoforms show markedly distinct regulatory properties that have not been fully explored. AC9 is highly expressed in vital organs such as the heart and the brain. Here, we report that the isoform-specific carboxyl-terminal domain (C2b) of AC9 inhibits the activation of the enzyme by Gs-coupled receptors (GsCR). In human embryonic kidney cells (HEK293) stably overexpressing AC9, cAMP production by AC9 induced upon the activation of endogenous β-adrenergic and prostanoid GsCRs was barely discernible. Cells expressing AC9 lacking the C2b domain showed a markedly enhanced cAMP response to GsCR. Subsequent studies of the response of AC9 mutants to the activation of GsCR revealed that residues 1268-1276 in the C2b domain were critical for auto-inhibition. Two main species of AC9 of 130 K and ≥ 170 K apparent molecular weight were observed on immunoblots of rodent and human myocardial membranes with NH-terminally directed anti-AC9 antibodies. The lower molecular weight AC9 band did not react with antibodies directed against the C2b domain. It was the predominant species of AC9 in rodent heart tissue and some of the human samples. There is a single gene for AC9 in vertebrates, moreover, amino acids 957-1353 of the COOH-terminus are encoded by a single exon with no apparent signs of mRNA splicing or editing making it highly unlikely that COOH-terminally truncated AC9 could arise through the processing or editing of mRNA. Thus, deductive reasoning leads to the suggestion that proteolytic cleavage of the C2b auto-inhibitory domain may govern the activation of AC9 by GsCR.

In the mammalian central nervous system (CNS) GABA receptors (GABARs) mediate neuronal inhibition and are important therapeutic targets. GABARs are composed of 5 subunits, drawn from 19 proteins, underpinning expression of 20-30 GABAR subtypes. In the CNS these isoforms are heterogeneously expressed and exhibit distinct physiological and pharmacological properties. We report the discovery of S44819, a novel tricyclic oxazolo-2,3-benzodiazepine-derivative, that selectively inhibits α5-subunit-containing GABARs (α5-GABARs). Current α5-GABAR inhibitors bind to the "benzodiazepine site". However, in HEK293 cells expressing recombinant α5-GABARs, S44819 had no effect on H-flumazenil binding, but displaced the GABAR agonist H-muscimol and competitively inhibited the GABA-induced responses. Importantly, we reveal that the α5-subunit selectivity is uniquely governed by amino acid residues within the α-subunit F-loop, a region associated with GABA binding. In mouse hippocampal CA1 neurons, S44819 enhanced long-term potentiation (LTP), blocked a tonic current mediated by extrasynaptic α5-GABARs, but had no effect on synaptic GABARs. In mouse thalamic neurons, S44819 had no effect on the tonic current mediated by δ-GABARs, or on synaptic (α1β2γ2) GABARs. In rats, S44819 enhanced object recognition memory and reversed scopolamine-induced impairment of working memory in the eight-arm radial maze. In conclusion, S44819 is a first in class compound that uniquely acts as a potent, competitive, selective antagonist of recombinant and native α5-GABARs. Consequently, S44819 enhances hippocampal synaptic plasticity and exhibits pro-cognitive efficacy. Given this profile, S44819 may improve cognitive function in neurodegenerative disorders and facilitate post-stroke recovery.

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The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.The bacterial surface has great importance in recognition events necessary to develop pathogen or symbiotic interactions as well as for bacteriophage attachment. In rhizobia, symbiotic nitrogen-fixing partners of different leguminous plants, exopolysaccharides, capsular polysaccharides, and lipopolysaccharides may play an essential role in infection (14). Recently it was shown that Sinorhizobium meliloti 41 produces a strainspecific capsular polysaccharide, the KR5 antigen, which is related to the group II K antigens from Escherichia coli. It can provide the same function as exopolysaccharides during symbiotic nodule development. In the S. meliloti 41 exoB background, the KR5 antigen is essential for the invasion of host plants (21,25,26).So far, three rkp gene clusters involved in biosynthesis of the KR5 antigen have been identified and shown to be important for the invasion of symbiotic hosts as well as for bacteriophage 16-3 infection. The rkp-1 region contains 10 genes (rkpA to rkpF) and appears to be involved in the production of a specific lipid carrier required for the biosynthesis of the KR5 antigen (12,18,21). The rkp-2 region harbors two genes (lpsL and rkpK), both required for lipopolysaccharide production; however, the second one (rkpK) encoding a UDP-glucose dehydrogenase, is also necessary for the synthesis of glucuronic acid, a precursor of the KR5 antigen (16). The rkp-3 region carries six strain-specific genes (rkpL to rkpQ) that are probably responsible for the synthesis of the pseudaminic acid component of the KR5 antigen. Additional genes have also been localized downstream from this cluster. Two of them (rkpZ and rkpY) affect capsular polysaccharide production, but three of them (rkpR and rkpTS) were identified only on the basis of their homology to genes involved in polysaccharide transport. Insertional mutations in these genes had no effect on the biosynthesis of KR5 antigen or on the development of symbiosis (17).In most cases, biochemical and immunological analy...

Rhizobial surface polysaccharides, including capsular polysaccharides (KPS), are involved in symbiotic infection. The rkp-3 locus of Sinorhizobium meliloti 41 is responsible for the production of pseudaminic acid, one of the components of the KR5 antigen, a strain-specific KPS. We have extended the sequence determination and genetic dissection of the rkp-3 region to clarify the structure and function of the rkpY gene and to identify additional rkp genes. Except for rkpY, no other genes were found where mutation affected the KPS structure and symbiosis. These mutants show a unique phenotype producing a low molecular weight polysaccharide (LMW PS). Creating double mutants, we have shown that biosynthesis genes of the KR5 antigen except rkpZ are not necessary for the production of this LMW PS. Polysaccharide analysis of genetically modified strains suggests that rkpY has pleiotropic effects on polysaccharide production. It directs KPS synthesis to the KR5 antigen and influences lipo-oligo 3-deoxy-d-manno-2 octulosonic acid (Kdo) production in S. meliloti 41. In addition, rkpY suppresses the lipo-oligoKdo production when it is introduced into S. meliloti 1021.